宽体金线蛭抗凝血活性寡肽研究
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摘要
目的
中药水蛭为水蛭科水蛭Hirudo nipponica Whitman,蚂蝗Whitmania pigra Whitman和柳叶蚂蝗Whitmania acranulata Whitman3种动物的干燥体,是活血逐瘀的常用中药,广泛用于多种心脑血管疾病,有较好疗效。其中,蚂蝗Hirudo nipponica Whitman即宽体金线蛭为我国临床主要应用品种。本文对宽体金线蛭中抗凝血活性成分的提取分离纯化工艺进行研究,并探讨宽体金线蛭抗凝血作用的物质基础。
方法
1.比较3种提取方法即干品水蛭水常温提取法、干品水蛭水加热提取法、干品水蛭石油醚脱脂后水加热提取法所得提取物的抗凝活性,并与鲜品水蛭的生理盐水提取法比较。同时比较了3种抗凝血活性检测方法即凝血酶滴定法、APTT试剂盒法及PT试剂盒法的效能,探讨宽体金线蛭抗凝血活性物质提取方法以及为本研究确定准确便捷的检测方法。
2.通过大鼠体外抗凝血实验,以PT、APTT、FIB及血浆粘度为检测指标,进一步观察通过最佳提取方法获得的粗提物的抗凝血活性。
3.运用凝胶色谱、离子交换色谱,分别采用了GE Sephadex G50Medium、Waters DEAE-cellulose52缓冲盐梯度洗脱、GE SephadexLH-20对上述粗提物进行分离。并采用液相色谱对所获得的活性部位进行纯化精制。
4.采用LC-MS测定活性单体分子量,并进行其抗凝血活性、理化性质的鉴定。
结果
1.3种提取方法均能获得具有较强的抗凝血活性的提取物。但干品水蛭石油醚脱脂后水加热提取法所得提取物抗凝血活性最好,且与干品冷提、干品热提具有统计学差异。3种抗凝血活性检测方法具有一致性结果,其中凝血酶滴定法对药典水蛭活量测定方法进行了改进,其实验现象易于观察,实验结果易于判断。
2.大鼠体外抗凝血实验证实干品水蛭石油醚脱脂后水加热提取法所获得粗提物能显著延长大鼠血浆PT、APTT,说明其可干扰内、外源性凝血因子的活性,而抑制凝血酶原向凝血酶的转化,使纤维蛋白原向纤维蛋白的转变过程受到抑制。同时其粗提物可以明显降低血浆FIB含量,对血浆粘度的影响不明显。
3.获得3个有较强抗凝血活性的活性部位,通过LC纯化精制,获得具有抗凝血活性的化合物单体。经理化鉴别为多肽类物质,其分子量分别为722.6、1880.6。结论
1.本研究发现加热并不破坏其抗凝血活性物质的提取,加石油醚脱脂提取能有效增加抗凝血活性物质的提取率,并减少颜色的干扰。
2.本研究提示凝血酶滴定法稳定、准确、便捷,可作为抗凝血类药物初步筛选中的首选方法,而APTT及PT试剂盒法等可作为进一步验证方法的检测体系。
3.本研究对《药典》记载凝血酶滴定法做了改进,将实验载体由试管改为白瓷板,并用解剖针不断搅拌监测,使实验终点清楚明确,该方法可以作为药典检测的常规方法。
4.本研究发现了宽体金线蛭具有抗凝血活性的寡肽,其分子量分别为722.954、1880.6,并初步认定分子量为1880.6的多肽为宽体金线蛭抗凝血的物质基础之一。在此基础上,将进一步针对分子量为1880.6的寡肽进行测序及合成、体内外活性研究,为宽体金线蛭抗凝血药物开发奠定基础。
Objective:
Traditional Chinese medicine leeches refer to dried bodies of three species of animals usually, including Hirudo nipponica Whitman, Whitmania pigra Whitman and Whitmania acranulata Whitman. Leechs are widely used in treatment of cardiovascular and cerebrovascular diseases by activating blood circulation to dissipate blood stasis, and the curative effect isobviously. Whitmania pigra Whitman is the main breed among them applied in clinical. To probe the anticoagulative material foundation of Whitmania pigra Whitmanarger in clinical, we studied the anticoagulant active components from Whitmania pigra Whitman.
Methods:
1We compared the anticoagulant activity of three different methods of extracting anticoagulant active components from Whitmanis pigra Whitmanwith physiological saline extraction method from fresh Whitma pigra. The extraction methods include unheated, heated extraction from dried Whitman pigra and heated extraction from skimmed dried Whitman pigra. We also evaluate three methods including the thrombin titration method, the APTT kit method and the PT kit method,which are applied to compare the anticoagulant activity and found out the most accuracy and speedy one.
2We tested the levels of PT, APTT, FIB and plasma viscosity by in vitro anticoagulant experiment in rats to evaluate the anticoagulant activity of the extracts using the best method.
3We separated the hirudo extraction by using Gel Chromatography and ion exchange chromatography which include GE Sephadex G50Medium water as eluting agent, DEAE-cellulose52Buffer salt as gradient elution, Sephadex G50desalination, GE SephadexLH-20water as eluting agent in order to study on anticoagulative material foundation, anticoagulant activity as a guidance.4We measured the molecular weights of the active monomer by LC-MS and tested the anticoagulant activity of the active monomer.
Result:
1The extracts of three extraction methods all have strong anticoagulant activity, while the heated extraction from skimmed dried Whitman pigra has the strongest anticoagulant activity, and has Statistically significant compared with unheated and heated extraction. the test conclusions by thrombin titration method, APTT kit method and PT kit method have the consistency, The improvement of thrombin titration method which is used to test leeches anticoagulant activity in Chinese Pharmacopoeia made the experimental phenomenology easily visible and the experimental result easy to evaluate.
2Experiment of leeches extraction in vitro in rats showed that it can extend PT and APTT, it prompte that leeches extraction can disturb internal blood coagulation factors and exogenous clotting factors, restrain prothrombin to thrombin, suppressed the transformation process of FIB to fibrin, significantly decreased FIB content in plasma, but the effect on plasma viscosity is not obvious.
3The experiments acquire three active sites which had strong anticoagulant activity, the activated monomers are gotten by using HPLC which is polypeptide on the basis of physical and chemical identification. The molecular weight are722.6and1880.6.
Conclusion:
1The anticoagulant activity of extraction from Whitmania pigra Whitman is not destroyed by heating. The extraction ratio of anticoagulant activity increases by degreased with ligarine and can reduce the distraction of color.
2The hrombin titration method may be used as a initial method to assess the anticoagulant activity, which is stable, accurate, convenient and easy to operate, while the APTT kit method and the PT kit methods may be applied as a further detect methods.
3The hrombin titration method in Chinese Pharmacopoeia, using the white porcelain plate as experimental carrier instead of test tube, monitoring by dissecting needle to make the experiment end point clea, which can be as conventional method in pharmacopoeia.
4The Whitmania pigra Whitman contains anticoagulant oligopeptides, whose molecular weights are722.954and1880.6. We suppose preliminarily that the heavier (MW=1880.6) oligopeptide is one of the anticoagulant activity material. The oligopeptide will be sequencing, synthesize, in vitro and in vivo activity study, lay the foundation for drug development in Whitmania pigra Whitman.
中药水蛭为水蛭科水蛭Hirudo nipponica Whitman,蚂蝗Whitmania pigra Whitman和柳叶蚂蝗Whitmania acranulata Whitman3种动物的干燥体,是活血逐瘀的常用中药,广泛用于多种心脑血管疾病,有较好疗效。其中,蚂蝗Hirudo nipponica Whitman即宽体金线蛭为我国临床主要应用品种。本文对宽体金线蛭中抗凝血活性成分的提取分离纯化工艺进行研究,并探讨宽体金线蛭抗凝血作用的物质基础。
方法
1.比较3种提取方法即干品水蛭水常温提取法、干品水蛭水加热提取法、干品水蛭石油醚脱脂后水加热提取法所得提取物的抗凝活性,并与鲜品水蛭的生理盐水提取法比较。同时比较了3种抗凝血活性检测方法即凝血酶滴定法、APTT试剂盒法及PT试剂盒法的效能,探讨宽体金线蛭抗凝血活性物质提取方法以及为本研究确定准确便捷的检测方法。
2.通过大鼠体外抗凝血实验,以PT、APTT、FIB及血浆粘度为检测指标,进一步观察通过最佳提取方法获得的粗提物的抗凝血活性。
3.运用凝胶色谱、离子交换色谱,分别采用了GE Sephadex G50Medium、Waters DEAE-cellulose52缓冲盐梯度洗脱、GE SephadexLH-20对上述粗提物进行分离。并采用液相色谱对所获得的活性部位进行纯化精制。
4.采用LC-MS测定活性单体分子量,并进行其抗凝血活性、理化性质的鉴定。
结果
1.3种提取方法均能获得具有较强的抗凝血活性的提取物。但干品水蛭石油醚脱脂后水加热提取法所得提取物抗凝血活性最好,且与干品冷提、干品热提具有统计学差异。3种抗凝血活性检测方法具有一致性结果,其中凝血酶滴定法对药典水蛭活量测定方法进行了改进,其实验现象易于观察,实验结果易于判断。
2.大鼠体外抗凝血实验证实干品水蛭石油醚脱脂后水加热提取法所获得粗提物能显著延长大鼠血浆PT、APTT,说明其可干扰内、外源性凝血因子的活性,而抑制凝血酶原向凝血酶的转化,使纤维蛋白原向纤维蛋白的转变过程受到抑制。同时其粗提物可以明显降低血浆FIB含量,对血浆粘度的影响不明显。
3.获得3个有较强抗凝血活性的活性部位,通过LC纯化精制,获得具有抗凝血活性的化合物单体。经理化鉴别为多肽类物质,其分子量分别为722.6、1880.6。结论
1.本研究发现加热并不破坏其抗凝血活性物质的提取,加石油醚脱脂提取能有效增加抗凝血活性物质的提取率,并减少颜色的干扰。
2.本研究提示凝血酶滴定法稳定、准确、便捷,可作为抗凝血类药物初步筛选中的首选方法,而APTT及PT试剂盒法等可作为进一步验证方法的检测体系。
3.本研究对《药典》记载凝血酶滴定法做了改进,将实验载体由试管改为白瓷板,并用解剖针不断搅拌监测,使实验终点清楚明确,该方法可以作为药典检测的常规方法。
4.本研究发现了宽体金线蛭具有抗凝血活性的寡肽,其分子量分别为722.954、1880.6,并初步认定分子量为1880.6的多肽为宽体金线蛭抗凝血的物质基础之一。在此基础上,将进一步针对分子量为1880.6的寡肽进行测序及合成、体内外活性研究,为宽体金线蛭抗凝血药物开发奠定基础。
Objective:
Traditional Chinese medicine leeches refer to dried bodies of three species of animals usually, including Hirudo nipponica Whitman, Whitmania pigra Whitman and Whitmania acranulata Whitman. Leechs are widely used in treatment of cardiovascular and cerebrovascular diseases by activating blood circulation to dissipate blood stasis, and the curative effect isobviously. Whitmania pigra Whitman is the main breed among them applied in clinical. To probe the anticoagulative material foundation of Whitmania pigra Whitmanarger in clinical, we studied the anticoagulant active components from Whitmania pigra Whitman.
Methods:
1We compared the anticoagulant activity of three different methods of extracting anticoagulant active components from Whitmanis pigra Whitmanwith physiological saline extraction method from fresh Whitma pigra. The extraction methods include unheated, heated extraction from dried Whitman pigra and heated extraction from skimmed dried Whitman pigra. We also evaluate three methods including the thrombin titration method, the APTT kit method and the PT kit method,which are applied to compare the anticoagulant activity and found out the most accuracy and speedy one.
2We tested the levels of PT, APTT, FIB and plasma viscosity by in vitro anticoagulant experiment in rats to evaluate the anticoagulant activity of the extracts using the best method.
3We separated the hirudo extraction by using Gel Chromatography and ion exchange chromatography which include GE Sephadex G50Medium water as eluting agent, DEAE-cellulose52Buffer salt as gradient elution, Sephadex G50desalination, GE SephadexLH-20water as eluting agent in order to study on anticoagulative material foundation, anticoagulant activity as a guidance.4We measured the molecular weights of the active monomer by LC-MS and tested the anticoagulant activity of the active monomer.
Result:
1The extracts of three extraction methods all have strong anticoagulant activity, while the heated extraction from skimmed dried Whitman pigra has the strongest anticoagulant activity, and has Statistically significant compared with unheated and heated extraction. the test conclusions by thrombin titration method, APTT kit method and PT kit method have the consistency, The improvement of thrombin titration method which is used to test leeches anticoagulant activity in Chinese Pharmacopoeia made the experimental phenomenology easily visible and the experimental result easy to evaluate.
2Experiment of leeches extraction in vitro in rats showed that it can extend PT and APTT, it prompte that leeches extraction can disturb internal blood coagulation factors and exogenous clotting factors, restrain prothrombin to thrombin, suppressed the transformation process of FIB to fibrin, significantly decreased FIB content in plasma, but the effect on plasma viscosity is not obvious.
3The experiments acquire three active sites which had strong anticoagulant activity, the activated monomers are gotten by using HPLC which is polypeptide on the basis of physical and chemical identification. The molecular weight are722.6and1880.6.
Conclusion:
1The anticoagulant activity of extraction from Whitmania pigra Whitman is not destroyed by heating. The extraction ratio of anticoagulant activity increases by degreased with ligarine and can reduce the distraction of color.
2The hrombin titration method may be used as a initial method to assess the anticoagulant activity, which is stable, accurate, convenient and easy to operate, while the APTT kit method and the PT kit methods may be applied as a further detect methods.
3The hrombin titration method in Chinese Pharmacopoeia, using the white porcelain plate as experimental carrier instead of test tube, monitoring by dissecting needle to make the experiment end point clea, which can be as conventional method in pharmacopoeia.
4The Whitmania pigra Whitman contains anticoagulant oligopeptides, whose molecular weights are722.954and1880.6. We suppose preliminarily that the heavier (MW=1880.6) oligopeptide is one of the anticoagulant activity material. The oligopeptide will be sequencing, synthesize, in vitro and in vivo activity study, lay the foundation for drug development in Whitmania pigra Whitman.
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